Synthesis and structure–activity relationship of benzetimide derivatives as human CXCR3 antagonists

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5819–5823

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Synthesis and structure–activity relationship of benzetimide derivatives as human CXCR3 antagonists Jean-Pierre Bongartz a,*, Mieke Buntinx a, Erwin Coesemans a, Bart Hermans a, Guy Van Lommen b, Jean Van Wauwe a a b

Johnson & Johnson PRD, RED EU, Turnhoutseweg 30, B-2340 Beerse, Belgium Galapagos NV, Generaal De Wittelaan L11 A3, 2800 Mechelen, Belgium

a r t i c l e

i n f o

Article history: Received 26 June 2008 Revised 25 July 2008 Accepted 25 July 2008 Available online 31 July 2008

a b s t r a c t The synthesis and evaluation of benzetimide derivatives showing potent CXCR3 antagonism are described. Optimization of the screening hits led to the identification of more potent CXCR3 antagonists devoid of anti-cholinergic activity and identification of the key pharmacophore moieties of the series. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: CXCR3 antagonist Benzetimide Chemokine

Cell migration is largely mediated by the interaction of some 50 secreted proteins, called chemokines, and 20 chemokine G proteincoupled receptors.1 They have been divided into 4 families according to the number and spacing of their conserved N-terminal cysteine residues (CC, CXC, CX3C, and XC). Chemokines and their receptors are also classified as constitutively expressed (homeostatic) or inducible/inflammatory (to control cell recruitment to sites of infection and inflammation). Human (h)CXCR3 is predominantly expressed on activated T helper 1(Th1) cells and activated by the interferon-inducible chemokines MIG (CXCL9), IP10 (CXCL10), and ITAC (CXCL11).2 Its function is mediated through Gai protein binding, leading to inhibition of cAMP formation and enhanced calcium mobilization, actin polymerization, and chemotaxis.3,4 Upregulation of hCXCR3 and its ligands, together with increased levels of disease-causing activated T cells in inflamed lesions, has been demonstrated in several chronic inflammatory diseases including organ transplant rejection,5 multiple sclerosis,6 colitis,7 insulitis,8 chronic obstructive pulmonary disease,9 allergic dermatitis,10 and rheumatoid arthritis.11 Taken together, these data indicate that inhibition of hCXCR3 activation provides a potential therapeutic intervention in a number of major Th1 cell-mediated inflammatory disorders. Recently, a number of experimental compounds directed to the CXCR3 target were reported.12 Herein we report the identification of a novel series

of hCXCR3 antagonists with promising activity and drug-like properties. Through screening of some 256,000 compounds for their inhibitory activity on cAMP production in ITAC-stimulated hCXCR3transfected CHO cells,13 a series of 3-disubstituted piperidine2,6-diones analogs of benzetimide (3) as novel hCXCR3 antagonists were identified as shown in Fig. 1. The activity of the most potent hit compound (1) was confirmed by assessing its ability to block the ITAC-activated GTPcS binding to hCXCR3-transfected CHO cell membranes,14 and compared to AMG 487 (2), the most advanced hCXCR3 antagonist at the time.15 As shown in Table 1, compound 1 inhibited the hCXCR3-mediated GTPcS binding with an IC50-value of 0.8 lM, comparable to that of AMG 487 (IC50 = 0.3 lM). The realization that 1 structurally resembles the muscarinic receptor antagonist benzetimide (3), prompted us to assess its potential anti-cholinergic activity. Confirming earlier data,17 the anti-

X

N

N N

X = Br 1 X=H 3

0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2008.07.115

N

N

F3C

* Corresponding author. Tel.: +32 14 60 22 45; fax: +32 14 60 57 55. E-mail address: [email protected] (J.-P. Bongartz).

O

O O H N O

O

O Amgen AMG 487 2

Figure 1. CXCR3 antagonists.

N

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Table 1 hCXCR3 antagonism and muscarinic binding data of compounds 1–3 and their enantiomers Compound

Chirality

hCXCR3 binding IC50a (lM)

1 1a 1b 2 3 3a 3b

±

0.78 0.61 1.33 0.33 >10 >10 >10

a b

+ ± +

Muscarinic receptor binding IC50b (lM) M1

M2

M3

9.6 <0.001 n.d.

>10 <0.001 n.d.

>10 <0.001 n.d.

>10 <0.001

>10 <0.001

>10 <0.001

GTPcS binding assay.14 Ref. 16 for assay.

Table 2 hCXCR3 antagonism: N-substitution on the 4-piperidine ring

O H N O R

N

Compounda

R

hCXCR3 binding IC50b (lM)

1 3 6 7 8 9 10 11 12 13

4-Br H 4-Cl 4-F 4-CH3 4-OMe 2,4-Cl 3,4-Cl 3-F-4-Cl 3-CF3-4-Cl

0.78 >10 0.83 4.57 2.40 4.27 3.16 1.26 0.34 2.51

a b

Racemic compounds, except for the ( )-isomer of compound 12. GTPcS binding assay.14

cholinergic activity of 3, measured by blocking the binding of Nmethylscopolamine to the human M1-3 muscarinic receptors, resides exclusively in its (+)-isomer, dexetimide (3b). By analogy, separation of 1 into its enantiomers 1a and 1b indicated that, although both stereoisomers produced comparable hCXCR3 antagonistic effects (Table 1), 1b possessed nanomolar activity against the muscarinic receptors, whereas 1a only showed a marginal activity toward the M1 receptor (IC50 = 9.6 lM). Moreover, submicromolar concentrations of 1a also reduced the ITAC-induced migration of activated human blood T lymphocytes (IC50 = 0.69 lM),18 and blocked the binding of [125I]ITAC to stimulated human T lymphocytes (IC50 = 0.33 lM).19 Additionally, when tested at 10 lM, 1a had no effect (<25% inhibition) on the binding of CHO cells expressing human CCR1, CCR2, CCR4, CXCR1/2, and CX3CR1 with [125I]-labeled MIP-1a, MCP-1, TARC, IL-8, and fractalkine, respectively.20 Reassured by these observations, we sought to identify more potent and selective hCXCR3 antagonists. Because of the pronounced difference in hCXCR3 binding potency between the para-bromo substituted benzyl-containing 1 (IC50 = 0.8 lM) and the unsubstituted benzyl analog 3, we first explored the contribution of N-substitution of the 4-piperidine ring. As shown in Table 2, the hCXCR3 antagonistic potency of 1 was found to be among the highest, and therefore we retained the parabromobenzyl substituent and concentrated our efforts on the glutarimide portion of the molecule. The synthesis of compounds 1, 3–50 is depicted in Scheme 1.21 The build-up of the dexetimide analogs required the base-catalyzed condensation of the benzyl-piperidone with the appropriate arylated acetonitriles. The alkene was reduced either catalytically or with NaBH4. The Michael addition with acrylonitrile promoted by Triton B was followed by the acid-mediated ring closure toward the glutarimide analogs 1, 3, and 6–24. Compound 21 containing the 2-OMe-5-sulfonic acid group was obtained in low overall yield (1.6% starting from acrylonitrile addition step b) as a side product

N

Y N

X

O

A A

a N

A

X= H or Br

N

O

H N

O

X

1,3 A A

d,e,f

6-24

NH

O

d, e, k, l

Y A

N O

m

N

N

N

X

X

4a X=H 4b X=Br

d

b,c,

N

g 25-29 f or Y = 3-COOH h 30 for Y = 3-CO OH i 31 for Y = 3-OMe j 32-37 for Y = H O N O

A A

A A

5a X=H 5b X=Br

Y A

Y A

R N

R N N

e,f

Br

38

39 R = COCH3 40 R = COOEt

41 R = COCH3 n,o 42 R = COOEt

43-50

Scheme 1. Reagents and conditions: (a) NaOMe, MeOH, reflux,18 h; (b) NaBH4, iPOH, reflux or 10%Pd/C, thiophene, H2, MeOH, rt; (c) acrylonitrile, Triton B, dry dioxane, 0 °C to rt, 18 h; (d) H2SO4/HOAc (1:4 v/v), 165 °C, 3–4 h, 10–40% for four steps; (e) 10% Pd/C, H2, MeOH, rt, >90%; (f) opt. substituted benzylhalide, triethylamine, DMF or CH2Cl2/ MeOH 1:1, rt, 18 h, variable yields; (g) SOCl2, CH2Cl2, rt, 4 days, then amines, rt; (h) (PhO)2P(O)N3, t-BuOH, 85 °C, 18 h then 10%TFA in CH2Cl2, rt, 18 h, 22% for two steps; (i) BBr3, CH2Cl2, rt, 18 h, 38%; (j) R-X, K2CO3, DMF, 60–70 °C, variable yields; (k) CBzCl, TEA, DMF, rt, 18 h, 71%; (l) BH3
THF 5 equiv, reflux, 6 h, 100%; (m) Ac2O, THF, rt, 18 h, 35% or ClCO2Et, triethylamine, CH2Cl2, rt, 18 h, 37%; (n) 48% HBr, reflux, 2 h, 70%; (o) 43: nBuOCHO, 100 °C, 4 h, 33%; 44: trifluoroacetic anhydride, THF, rt, 4 days, 37%; 45, 46: RCOCl, triethylamine, CH2Cl2, rt, 65–77%; 47: ClCH2CONH2, triethylamine, CH2Cl2, rt, 18 h, 49%; 48: PheNCO, CH2Cl2, rt, 20 h, 64%; 49: Me3SiNCO, dry dioxane, 90 °C, 20 h, 14%; 50: NH2SO2NH2, pyridine, 120 °C, 18 h, 16%.

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O O

N

N

N a

N b ,c

H N

HN

H N

N

Od

Y

Br

51

53

52

N

f

H O N

g, h N

N

i, d Br

O O 56

N

54 Y = O e 55 Y = H H O N

58

57

Scheme 2. Reagents and conditions: (a) methylacrylate, NaOMe, xylene, 65 °C, 4–6 h, 92%; (b) 10% Pd/C, H2, MeOH, rt; (c) Raney nickel, H2, MeOH, rt, 56% for two steps; (d) pBr benzylchloride, triethylamine, DMF, rt, 18 h, 40–45%; (e) BH3
THF, reflux, 18 h, 94%; (f) diethylcarbonate, LDA, THF, 78 °C, 40 min to rt, 2 h; (g) acrylonitrile, Triton B, dry dioxane, rt,18 h; (h) Raney nickel, H2, THF, rt, 25% for three steps; (i) Pd/C, H2, MeOH, 75 °C, 80%.

during an attempt to synthesize the 2-methoxy substituted variant. Acid chloride activation of the acid 24 enabled the formation of amides 25–29. The two-step Curtius rearrangement of acid 24 yielded the aniline 30. BBr3 mediated demethylation of 20 yielded the phenol 31. Base-promoted alkylation of the imide 1 produced 32–37. The borane reduction of the imide did not proceed on the benzyl-protected piperidine derivatives, but was quantitative on the CBz-protected intermediate leading to intermediate 38. Further coupling and deprotection steps produced the required compounds 43–50. The lactam analogs 54, 58 of the lead compounds were synthesized as described in Scheme 2.21 A Michael addition of methylacrylate on intermediate 51 was followed by the one-pot, two-step Pd-mediated debenzylation and the Raney nickel reduction of the nitrile to the amine, which spontaneously ring closed to lactam 53. Coupling of p-bromobenzylchloride gave 54 and lactam reduction produced 55. The reversed lactam 58 required the LDA-mediated carboxyethylation of commercially available 4-benzylpyridine with diethylcarbonate toward 56. Michael addition of acrylonitrile and Raney nickel-mediated reduction of the nitrile produced the lactam 57. Catalytic reduction of the pyridine to piperidine, followed by coupling of p-bromobenzylchloride yielded 58.

Table 4 hCXCR3 antagonism: modification of the glutarimide moiety

Table 3 hCXCR3 antagonism: modification of the phenyl moiety

O

Br N R

H N

O

R

hCXCR3 binding IC50a (lM)

1 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

C 2-Pyridyl 3-Pyridyl 4-Pyridyl C C C C C C C C C C C C C C C

H H H H 4-F 2,4-F 4-OMe 3-OMe 2-OMe-5-SO3H 3-Cl 4-CO2H 3-CO2H 3-CONH2 3-CONHMe 3-CONH(CH2)3OH 3-CO-Morpholine 3-CO-N-Me-piperazine 3-NH2 3-OH

0.78 0.50 1.00 0.63 1.26 0.12 3.63 1.55 0.17 1.45 5.01 0.63 3.16 3.16 1.26 0.79 1.26 0.32 1.00

R N

Y

N

A AA

A

GTPcS binding assay.14

X

Br

Compound

a

As illustrated in Table 3, replacement of the unsubstituted phenyl in 1 by 2-, 3-, or 4-pyridine (compounds 14–16) was neutral in terms of receptor potency. The para-position accepts preferably a small substituent such as fluorine in 17 instead of the larger methoxy 19 and carboxylic acid 23. The meta-position tolerates a variety of substituents of different sizes and polarity such as exemplified by 20, 22, and 24–31. Introduction of solubility enhancing groups as in 24, 27, and 29 at this position is tolerated for activity and could be a good starting point for further optimization of the drug-like properties of this series. The 7-fold potency gain seen for 2,4-fluor analog 18 relative to the 4-fluor analog 17 and the high activity of 21 suggest that an ortho substitution is beneficial for activity. The contribution of the glutarimide group is shown in Table 4. The imide hydrogen was substituted by various alkyl groups as in 32–37 without improvement in potency. Retaining one (54 and 58) of the carbonyl functions in the glutarimide ring is essential for high activity. The conversion of the glutarimide into a 3-piperidine ring (55) results in a decrease of activity, unless the N-piperidine atom is, for instance, substituted with a carbonyl-containing residue such as in 41–49. Sulfonamide 50 is equipotent to the ureum 49. Highest potency in this series (IC50 = 0.06–0.08 lM) was seen

Compound

X

Y

R

hCXCR3 binding IC50a (lM)

1 32 33 34 35 36 37 41 42 43 44 45 46 47 48 49 50 54 55 58

O O O O O O O H H H H H H H H H H H H O

O O O O O O O H H H H H H H H H H O H H

H Me Et Pr i-Pr CH2CH2OCH3 CH2SCH3 Acetyl Ethylcarbamate Formyl Trifluoroacetyl Propionyl Cyclopropionyl a-Acetamide Phenylureum Ureum Sulfonamide H H H

0.78 2.51 2.51 5.01 5.01 2.00 1.00 0.14 0.32 0.15 0.69 0.29 0.18 0.08 0.06 0.25 0.21 0.50 >10 1.58

a

GTPcS binding assay.14

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Table 5 hCXCR3 and muscarinic receptor antagonism: enantiomeric effect

X

Br

R2 N Y

N R1 Compound

Chirality

X

Y

R1

R2

hCXCR3 binding IC50a (lM)

1 1a 1b 18 18a 18b 41 41a 41b 47 47a 47b 48 48a 48b

±

O O O O O O H H H H H H H H H

O O O O O O H H H H H H H H H

H H H 2,4-F 2,4-F 2,4-F H H H H H H H H H

H H H H H H Acetyl Acetyl Acetyl a-Acetamide a-Acetamide a-Acetamide Phenylureum Phenylureum Phenylureum

0.78 0.61 1.33 0.12 0.06 0.33 0.14 0.11 2.31 0.08 0.81 0.06 0.06 0.03 6.31

a b

+ ± + ± + ± + ± +

Muscarinic receptor binding IC50b (lM) M1

M2

M3

9.6 <0.001

>10 <0.001

>10 <0.001

>10 <0.001

>10 <0.001

>10 <0.001

10 >10

10 >10

3 >10

5 >10

2 >10

1.1 >10

>10 8.9

>10 >10

>10 >10

GTPcS binding assay.14 Ref. 16 for assay.

with the a-acetamide (47) and the phenylureum (48) substituents, but at the cost of less favorable PK properties in the case of 48 (data not shown). Table 5 lists the CXCR3 and muscarinic receptor binding activity of our most potent racemic compounds and their enantiomers. The enantiomers were separated by chiral HPLC or SFC and the enantiomers were distinguished by their optical rotations (levo or dextro rotatory), without further determination of their absolute configuration. The muscarinic activity of the opposite enantiomer of the hit 1a lead us to select the enantiomeric pairs of those compounds for screening on a panel of muscarinic receptors in a radioligand binding assay.16 The isomers of compounds 1 and 18, which share the same benzetimide (3) scaffold, display comparable hCXCR3 antagonistic activities, while their anti-muscarinic activity is almost totally confined to the (+)-isomer form. However, N-substitution of the 3-piperidine ring with carbonyl-containing functions (compounds 41, 47, and 48) indicated that their optical isomers produced weak or no anti-muscarinic activity and that strong CXCR3 antagonistic activity was largely confined to a single enantiomer 41a, 47b, and 48a. Of note, although not tested on a regular basis, our compounds showed comparable potencies against mouse CXCR3 (data not shown), excluding issues of speciation that are often encountered in the search for chemokine receptor antagonists. Taken together, we have identified a novel series of 3-piperidine compounds that produce submicromolar antagonistic activity against human and murine CXCR3. The compounds were optimized from the HTS hit 1, a structural analog of the anti-cholinergic benzetimide. From enantiomeric separation of the most potent 3-piperidine compounds it was learnt that CXCR3 antagonism and anti-cholinergic effects were not linked. In vivo experiments are underway to assess the potential of these compounds as antiinflammatory agents. References and notes 1. Murphy, P. M.; Baggiolini, M.; Charo, I. F.; Horuk, R.; Matsushima, K.; Miller, L. H.; Oppenheimer, J. J.; Power, C. A. Pharmacol. Rev. 2000, 52, 145. 2. Loetscher, M.; Loetscher, P.; Brass, N.; Meese, E.; Moser, B. Eur. J. Immunol. 1998, 28, 3696. 3. Medina, J.; Johnson, M. G.; Collins, T. L. Ann. Rep. Med. Chem. 2005, 40, 215.

4. Tensen, C. P.; Flier, J.; Van Der Raaij-Helmer, E. M.; Sampat-Sardjoepersad, S.; Van Der Schors, R. C.; Leurs, R.; Scheper, R. J.; Boorsma, D. M.; Willemze, R. J. J. Invest. Dermatol. 1999, 112, 716. 5. (a) Lazzeri, E.; Romagnani, P. Curr. Drug Targets 2005, 5, 109; (b) Hancock, W.; Lu, B.; Gao, W.; Csizmadia, V.; Faia, K.; King, J. A.; Smiley, S. T.; Ling, M.; Gerard, N. P.; Gerard, C. J. Exp. Med. 2000, 192, 1515; (c) Belperio, J. A.; Keane, M. P.; Burdick, M. D.; Lynch, J. P., III; Zisman, D. A.; Xue, Y. Y.; Li, K.; Ardehali, A.; Ross, D. J.; Strieter, R. M. J. Immunol. 2003, 171, 4844. 6. (a) Sørensen, T. L.; Trebst, C.; Kivisäll, P.; Klaege, K. L.; Majmudar, A.; Ravid, R.; Lassmann, H. J. Neuroimmunol. 2002, 127, 59; (b) Sindern, E.; Patzhold, T.; Ossege, L. M.; Gisevius, A.; Malin, J.-P. J. Neuroimmunol. 2002, 131, 186. 7. (a) Olsen, D. B.; Strieter, R. M.; Ransohoff, R. M.; Sellebjerg, F. J. Neuroimmunol. 2002, 127, 59; (b) Sasaki, S.; Yoneyama, H.; Suzuki, K.; Suriki, H.; Aiba, T.; Watanabe, S.; Kawachi, H.; Shimizu, F.; Matsuashima, K.; Asakurri, H.; Narumi, S. Eur. J. Immunol. 2002, 32, 3197. 8. Frigerio, S.; Junt, T.; Lu, B.; Gerard, C.; Zumsted, U.; Holländer, G. A.; Piali, L. Nat. Med. 2002, 8, 1414. 9. Saetta, M.; Mariani, M.; Panina-Bordignon, P.; Turato, G.; Buonsanti, C.; Baraldo, S.; Bellettato, C. M.; Papi, A.; Corbetta, L.; Zuin, R.; Sinigaglia, F.; Fabbri, L. M. Am. J. Respir. Crit. Care 2002, 165, 1404. 10. Flier, J.; Boorsma, D. M.; Bruynzeel, D. P.; van Beek, P.; Stoof, T. J.; Scheper, R. J.; Willemze, R.; Tensen, C. P. J. Invest. Dematol. 1999, 113, 574. 11. Qin, S.; Rottman, J. B.; Myers, P.; Kassam, N.; Wienblatt, M.; Loetscher, M.; Koch, A. E.; Moser, B.; Mackay, C. R. J. Clin. Invest. 1998, 101, 746. 12. (a) Storelli, S.; Verdijk, P.; Verzijl, D.; Timmerman, H.; van de Stolpe, A.; Tensen, C. P.; Smit, M. J.; De Esch, I. J. P.; Leurs, R. Bioorg. Med. Chem. Lett. 2005, 15, 2910; (b) Allen, D. R.; Bolt, A.; Chapman, G. A.; Knight, R. L.; Meissner, J. W. G.; Owen, D. A.; Watson, R. J. Bioorg. Med. Chem. Lett. 2007, 17, 697; (c) Johnson, M.; Li, A.-R.; Liu, J.; Fu, Z.; Zhu, L.; Miao, S.; Wang, X.; Xu, Q.; Huang, A.; Marcus, A.; Xu, F.; Ebsworth, K.; Sablan, E.; Danao, J.; Kumer, J.; Dairaghi, D.; Lawrence, C.; Sullivan, T.; Tonn, G.; Schall, T.; Collins, T.; Medina, J. Bioorg. Med. Chem. Lett. 2007, 17, 3339; (d) Watson, R. J.; Allen, D. R.; Birch, H. L.; Chapman, G. A.; Galvin, F. C.; Jopling, L. A.; Knight, R. L.; Meier, D.; Oliver, K.; Meissner, J. W. G.; Owen, D. A.; Thomas, E. J.; Tremayne, N.; Williams, S. C. Bioorg. Med. Chem. Lett. 2008, 18, 147; (e) Du, X.; Chen, X.; Mihalic, J. T.; Deignan, J.; Duquette, J.; Li, A.R.; Lemon, B.; Ma, J.; Miao, S.; Ebsworth, K.; Sullivan, T. J.; Tonn, G.; Collins, T. L.; Medina, J. C. Bioorg. Med. Chem. Lett. 2008, 18, 608; (f) Li, A. R.; Johnson, M. G.; Liu, J.; Chen, X.; Du, X.; Mihalic, J. T.; Deignan, J.; Gustin, D. J.; Duquette, J.; Fu, Z.; Zhu, L.; Marcus, A. P.; Bergeron, P.; McGee, L. R.; Danao, J.; Lemon, B.; Carabeo, T.; Sullivan, T.; Ma, J.; Tang, L.; Tonn, G.; Collins, T. L.; Medina, J. C. Bioorg. Med. Chem. Lett. 2008, 18, 688. 13. Stably transfected hCXCR3-expressing CHO cells were suspended at 106 cells/ well in DMEM/HAM’S F12 medium supplemented with 10% fetal bovine serum and 1 mM isobutylmethylxanthine. After a 60-min pre-incubation at room temperature with compound, forskolin (30 lM) and ITAC (0.3 lM, Peprotech) were added. After 30 min, intracellular cAMP was measured using the cAMP dynamic kit from CIS bio International. 14. Membranes of hCXCR3-expressing CHO cells were diluted in incubation buffer (20 mM Hepes, 100 mM NaCl, 3 lM guanine diphosphate, 1 mM MgCl2, pH 7.4, 14 lg/ml saponin) and pre-incubated with compound for 30 min at 30 °C in 96-well flashplates (Perkin-Elmer), before stimulation with human ITAC (3 nM) or IP-10 (150 nM) (R&D Systems). [35 S]GTPcS (0.25 nM, 1119 Ci/

J.-P. Bongartz et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5819–5823

15.

16. 17.

18.

mmol, Amersham) was then added and, 30 min later, bound radioactivity was determined on a Topcount (Packard). (a) Johnson, M.G.; Li, A.; Liu, J.; Marcus, A.P.; Huang, A.X.; Medina, J.C. Abstracts of Papers; 231st National Meeting of the American Chemical Society: Atlanta, GA, 2006.; (b) Johnson, M.; Li, A. R.; Liu, J.; Fu, Z.; Zhu, L.; Miao, S.; Wang, X.; Xu, Q.; Huang, A.; Marcus, A.; Xu, F.; Ebsworth, K.; Sablan, E.; Danao, J.; Kumer, J.; Dairaghi, D.; Lawrence, C.; Sullivan, T.; Tonn, G.; Schall, T.; Collins, T.; Medina, J. Bioorg. Med. Chem. Lett. 2007, 17, 3339. Muscarinic receptor binding data were obtained from MDS Pharma Services, Bothell, WA 98021, USA. Janssen, P. A. J.; Niemegeers, C. J. E.; Schellekens, K. H. L.; Demoen, P.; Lenaerts, F. M.; Van Nueten, J. M.; Van Wijngaarden, I.; Brugmans J. Drug Res. 1971, 21, 1365. Human peripheral blood mononuclear cells (PBMCs) were activated with PHA and human IL-2 (R&D Systems) for 3 days and further cultured with IL-2 until the day of analysis (days 9–14). Cells were labeled with Calcein-AM (Invitrogen) and dissolved in HBSS supplemented with 0.2% BSA. After pre-

5823

incubation with compound (10 min, 25 °C), 20 ll cell suspension (80,000 cells) was added in 6-fold to the topside of the filter, while the bottom wells of the 96-well chemotaxis chambers (ChemoTx, NeuroProbe) were filled with 3 nM human ITAC. After 3 h at 37 °C, non-migrated cells were removed from the filter and migrated cells were measured using a fluorescent plate reader. 19. PHA/IL 2-activated human PBMC (prepared as above; 2.5  105/well) were dissolved in binding buffer (50 mM Hepes, 5 mM MgCl2, 1 mM CaCl2, 0.02% NaN3, 0.5% BSA, pH 7.4) and pre-incubated with compound for 30 min, before 80 pM [125I]-ITAC (2000 Ci/mmol, Amersham) was added and kept for 60 min at 4 °C. Cells were harvested upon GF/B filter plates (presoaked in 0.5% polyethyleneimine) and radioactivity was determined by liquid scintillation counting. 20. Chemokine receptor binding data were obtained from MDS Pharma Services, Bothell, WA 98021, USA. 21. a Coesemans, E.; Bongartz, J.-P. A. M.; Van Lommen, G. R. E.; Van Wauwe, J. P. F.; Buntinx, M. WO Patent 090826, 2007.; b Coesemans, E.; Bongartz, J.-P. A. M.; Van Lommen, G. R. E. WO Patent 090836, 2007.